TSEs are caused by a group of related infectious agents that produce neurodegenerative disease in many species. Some agents, such as BSE, have increased in virulence with broad epidemic spread worldwide. These infectious agents can provoke many innate immune responses at very early asymptomatic stages, and only later does one find secondary patholgocial changes in prion protein (PrP). This, as well the existence of many different strains of agent, point to a causal virus with non-host coding sequences. Previous research on our CJD animal models revealed 25nm virus like particles in highly infectious brain fractions. To better resolve the fundamental structure and composition of TSE infectious particles in-situ, and to elucidate novel biological features of different strains, we transmitted various TSE agents to cultured cells. In these highly infectious cells, that show no neurodegenerative changes, we identified 25nm particles in ordered arrays that are characteristic for viruses. They do not contain PrP, and hence are not prions. These particle arrays, moreover, correspond well with those found in a variety of infected brains, as well as with the isolated 25nm particles in brain fractions. The TSE-specific virus like arrays were identified in two different cell types infected with two very different TSE agents (FU-CJD and 22L-scrapie). We propose to develop rapid live cell culture assays of infectivity for these two TSE agents. This rapid assay of infectivity will be verified by parallel animal titrations. Currently, assays of TSE infectious agents involve long and expensive animal titrations, and a rapid cell assay is critical for improved purifications of infectious particles from secondary pathological products. We will compare cell and animal assays to understand the respective limitations and advantages of each, and then use them for fundamental studies. Increasingly purified particles, and their molecular components, will be applied to susceptible cells to demonstrate which specific structures or components are the causal infectious pathogen by Koch's principles. The isolation of highly infectious particles and the definition of their molecular components can lead to sensitive new diagnostic screening methods for preventing further spread of TSE diseases, and possibly to a vaccine with non-host TSE components. Rapid infectivity assays can also benefit the design of drugs to ambush the progressive replication of these agents before irreversible neurodegeneration begins.